Compositions and methods for cancer prevention and treatment derived from Inula britannica

ABSTRACT

Compositions and methods for preventing and treating cancer are provided that comprise extracts of  Inula britannica  or compounds isolated therefrom.

[0001] This application is a continuation of U.S. application Ser. No.10/079,038, filed Feb. 20, 2002, which claims the benefit of U.S.provisional application Ser. No. 60/270,189, filed Feb. 21, 2001.

BACKGROUND OF THE INVENTION

[0002]Inula britannica is a traditional Chinese medicinal herb that hasbeen used to treat bronchitis and inflammation. A variety of this plant,known as Inula britannica chinensis has been used as an insecticide incertain areas of China. Both Inula britannica and Inula britannicachinensis have been examined in order to determine the chemicalconstituents responsible for its pharmacological effects. A variety ofsesquiterpenes have been isolated. In the case of Inula britannicachinensis, three specific sesquiterpene lactones were identified,including britannilactone, 1-O-acetylbritannilactone, and1,6-O,O-diacetylbritannilactone (Zhou, B-N. et al. 1993. Phytochemistry34:249-252).

[0003] Some natural plant extracts have been shown to have activity aschemopreventive agents. An example, taxol, acts by inducing Bcl-2phosphorylation in cancer cells which leads to programmed cell death(Haldar, S. Et al. 1996. Cancer Res. 56:1253-1255). The Bcl-2 protein isa member of a family of cytoplasmic proteins which regulates cell death.Bcl-2 has been shown to promote cell survival by inhibiting the processof cell death known as apoptosis. Whereas Bcl-2 acts to inhibitapoptosis, Bax, another cytoplasmic protein, counteracts this protectiveeffect; Bcl-2 is also thought to protect cells from apoptosis bydimerizing with Bax (Hunter, J. J. et al. 1996. J. Biol. Chem.271:8521-8524). The phosphorylation of Bcl-2 interferes with thehomodimers and subsequent apoptosis (Haldar, S. et al. 1995. Proc. Natl.Acad. Sci. USA 92:4507-4511; Haldar, S. Et al. 1996. Cancer Res.56:1253-1255). Therefore, therapeutic strategies to inactivate Bcl-2 arebeing sought as a way to improve clinical results with certain drugs. Ithas now been found that an extract of Inula britannica has activityrelevant to prevention and treatment of cancer due to its activity tophosphorylate Bcl-2.

SUMMARY OF THE INVENTION

[0004] An object of the present invention is a composition that inducesapoptosis in cells which comprises an extract of Inula Britannica orcompounds isolated therefrom. In a preferred embodiment the compositioncomprises sesquiterpene lactone.

[0005] Another object of the present invention is a method for inducingapoptosis in cells comprising contacting cells with an extract of InulaBritannica or compounds isolated therefrom so that apoptosis is induced.

[0006] Yet another object of the present invention is a method forpreventing and treating cancer which comprises administering aneffective amount of extract of Inula Britannica or compounds isolatedtherefrom.

DETAILED DESCRIPTION OF THE INVENTION

[0007] An extract of Inula britannica has been isolated that has use asa cancer preventative agent due to its activity to induce apoptosis incancer cells. The extract contains several sesquiterpene compounds,including but not limited to the sesquiterpene lactones known asbritannilactone, 1-O-acetylbritannilactone (OABL), and1,6-O,O-diacetylbritannilactone (OODABL). The extract and the chemicalsisolated therefrom can be used as a pharmaceutical for cancer treatmentand/or prevention as well as a medical food, or nutraceutical, and adietary supplement.

[0008] The flowers (approximately 10 kg) of Inula britannica var.chinensis were extracted three times with 95% ethanol. Thechloroform-soluble fraction of the ethanol extract (500 g) waschromatographed on a silica gel column eluting with a gradient ofchloroform-methanol. From the fraction collected withchloroform-methanol (20:1), 1,6-O,O-diacetylbritannilactone (52 g) wasobtained. From the fraction collected with chloroform-methanol (10:1),1-O-acetylbritannilactone (10.5 g) was obtained.

[0009] Experiments were performed to determine the activity of two ofthe sesquiterpene compounds isolated from Inula britannica, OABL andOODABL, as cancer preventative agents. Using a Western blot technique,the ability of these compounds to phosphorylate Bcl-2 in cancer cellswas examined. Using the breast cancer cell line MCF-7, it was found thatOABL induced Bcl-2 phosphorylation, with effective doses of 10 and 20μM. OODABL induced Bcl-2 phosphorylation at lower effective doses, 2.5and 5 μM. These results were compared to the ability of a knownchemotherapeutic paclitaxel, which induced Bcl-2 phosphorylation at adose of 100 μM.

[0010] These data indicate that the sesquiterpenes are more potent thanpaclitaxel at inducing Bcl-2 phosphorylation in MCF-7 cells.

[0011] In two ovarian cancer cell lines, OVCAR and PA-1, similar resultswere seen. In OVCAR cells, OODABL induced Bcl-2 phosphorylation at adose of 5 μM, while OABL induced Bcl-2 phosphorylation at a dose of 10μM. In PA-1 cells, OODABL induced Bcl-2 phosphorylation at a dose of 5μM, while OABL induced Bcl-2 phosphorylation at a dose of 10 μM.

[0012] In a prostate cancer cell line, these compounds were also able toinduce Bcl-2 phosphorylation. OODABL induced Bcl-2 phosphorylation at adose of 5 μM while OABL induced Bcl-2 phosphorylation at a dose of 10μM.

[0013] Using cleavage of PARP as an indicator of apoptosis, thecompositions of the present invention were tested in PA-1 ovary cells.PARP is a 116 kD nuclear poly (ADP-ribose)polymerase that appears to beinvolved in DNA-repair, predominantly in response to environmentalstress (Satoh, M. S. and T. Lindahl. 1992. Nature 356:356-358). PARP isimportant for cells to maintain their viability. Cleavage of PARPfacilitates cellular disassembly and serves as a marker of cellsundergoing apoptosis (Oliver, F. J. et al. 1998. J. Biol. Chem.273:33533-33539). This protein can be cleaved by many ICE caspases toform a 85 kD protein in cells undergoing apoptosis. PA-1 cells weretreated with OODABL for 24 hours. Cells were lysed and PARP cleavage wasmeasured by Western Blot using a monoclonal antibody to PARP(Pharmingen, Inc., San Dieago, Calif.). OABL induced PARP cleavage atdoses of 10 and 20 μM, while OODABL induced PARP cleavage at a dose of 5μM.

[0014] The effect of OODABL on cell cycle was analyzed by flowcytometery using breast cancer cell lin T47D. OODABL arrested cells atthe G2/M phase at a 20 μM concentration as compared to control cells.

[0015] The effect of OODABL on microtubules was examined by indirectimmunofluorescence of MCF-7 cells using an antibody to tubulin after 12hours treatment with either a vehicle control, 10 μM paclitaxel(positive control), or 20 μM OODABL. The results showed, that OODABLpolymerized microtubules like paclitaxel.

[0016] In a TUNEL assay, the effect of OODABL on late apoptosis wasexamined. HL-60 cells lines were subjected to flow cytometry analysisusing APO-BRDU. Apoptosis was detected by incorporation of Br-dUTP usinga fluorescein labeled anti-BrdU monoclonal antibody after treatment witha vehicle control, 1 μM camptothecin or 20 μM OODABL for 12 hours.OODABL was shown to induce apoptosis, as did the positive controlcamptothecin.

[0017] Cell viability was then assessed in a microculturetetrazolium/formazan assay (MTT; Scudiero, D. A. et al. 1988. CancerRes. 48:4827-4833) using a variety of cell lines. Absorbance wasmeasured at 550 nm and cell viability was expressed as the percentage ofdrug treated cells relative to that of controls. The IC₅₀ was thendefined as the concentration of drug that produced a 50% decrease incell viability relative to controls. OODABL was first tested in avariety of cell lines. Results in MCF-7 cells treated with variousconcentrations of OODABL (1.25, 12.5, 25.50 and 100 μM OODABL) showedthat cell viability decreased with treatment in a dose-dependent manner.The IC₅₀ was less than 12.5 μM. In PA-1 cells treated with variousconcentrations of OODABL (1.953, 3.9, 7.815, 15.625, 31.25, and 62.5 μMOODABL), cell viability was decreased in a dose-dependent manner with anIC₅₀ of less than 7.815 μM. In DU-145 cells treated with variousconcentrations of OODABL (3.4, 7.86, 15.6, 31.5, 62.5, and 125 μMOODABL), cell viability was decreased in a dose-dependent manner with anIC₅₀ of less than 15.6 μM. In NCI-H-460 cell treated with variousconcentrations of OODABL (3.9, 7.81, 1'5.62, 31.25, 62.5 and 125 μMOODABL), cell viability was decreased in a dose-dependent manner with anIC₅₀ of between 31.25 and 62.5 μM. In NIH 3T3 (normal mouse fibroblasts)cells treated with various concentrations of OODABL (1, 10, 20 and 50 μMOODABL), cell viability was decreased in a dose-dependent manner with anIC₅₀ of 50 μM.

[0018] OABL was then tested in some of these same cell lines. In MCF-7cells, OABL was tested at doses of 0.3 nm, 3 nm, 30 nm, 300 nm, 3 μM,and 30 μM. Results showed that OABL decreased cell viability with anIC₅₀ of around 200 μM. In PA-1 cells, OABL (1.953, 3.9, 7.815, 15.62,31.25 and 62.5 μM) decreased cell viability with an IC₅₀ of about 2 μM.In Du-145 cells, OABL (4.68, 9.37, 18.75, 37.5, 75 and 100 μM) decreasedcell viability.

[0019] Cell cytotoxicity was also assessed by a clonogenic assay. MCF-7breast cells were treated with various concentrations of OODABL (625 nm,1.25, 2.5, 5 and 10 μM) for 15 days and cells were then stained withmethylene blue and colonies counted. The IC₅₀ was in the range of 2.5 to5 μM OODABL. PC-3 prostate cells were treated with variousconcentrations of OODABL (20 and 200 μm, and 2 and 20 μM) for 15 daysand cells were then stained with methylene blue and colonies counted.The IC₅₀ was in the range of 200 nm OODABL. RKO cells were treated withvarious concentrations of OODABL (20 and 200 nm, and 2 and 20 μM) for 15days and cells were then stained with methylene blue and coloniescounted. The IC₅₀ was in the range of 20 μM OODABL. Baby rat kidneycells were transformed with E1A and transfected with the Bcl-2 gene toform BRK-4B-Bcl-2 cells. These cells were treated with variousconcentrations of OODABL (20 and 200 nm, and 2, 10 and 20 μM) for 15days and cells were then stained with methylene blue and coloniescounted. The IC₅₀ was in the range of 200 nM OODABL. Baby rat kidneycells were transformed with E1A and transfected with Bcl-2 gene in whichphosphorylation sites were mutated to form phosphomutant BRK-4B-Bcl-2cells. These cells were treated with various concentrations of OODABL(20 and 200 nm, and 2 and 20 μM) for 15 days and cells were then stainedwith methylene blue and colonies counted. The IC₅₀ was in the range of 2μM.

[0020] The level of Bcl-2 phosphorylation was then assessed in thenon-mutated and mutated BRK-4B-Bcl-2 cells using a Western blot assay.Cells were initially treated for 12 hours with the test compound, OODABL(at concentrations of 10, 20, 30, 40, or 60 μM). Taxol was used as apositive control at a concentration of 5 μM. Cells were then lysed inice cold radio-immune precipitation buffer with inhibitors. Equivalentamounts of proteins were electrophoresed by 12% dodecylsulfate-polyacrylamide gel electrophoresis and transferred tonitrocellulose. Bcl-2 and phosphorylated Bcl-2 proteins were detectedusing a monoclonal Bcl-2 primary antibody and a secondary goatanti-mouse horseradish peroxidase conjugated antibody followed byenhanced chemiluminescence detection. The results showed that there wasa dose-dependent increase in Bcl-2 phosphorylation with OODABL in thenon-mutated cells. Taxol also produced an increased in proteinphosphorylation. In the mutated rat kidney cells, there was nophosphorylation evident with either taxol or OODABL.

[0021] These data demonstrate that the extract of Inula britannica hasuse as a cancer preventative and treatment agent due to its activity toinduce apoptosis and cell cytotoxicity in cancer cells. The extract andthe chemicals isolated therefrom, OABL and OODABL, can be used as apharmaceutical for cancer treatment and/or prevention as well as amedical food, or nutraceutical, and a dietary supplement.

[0022] The data presented support the development of either foods foranimal consumption, where animals include humans, or as dietarysupplements for animals including humans. These foods and supplementsare referred to by those of skill in the art as “nutraceuticals”.Compositions of the present invention would be useful as nutraceuticalsfor prevention or treatment of cancer. One of skill would be able to usethe results of experiments in cells and animals to determine aneffective amount to be administered in humans. An effective amount wouldbe an amount that induces apoptosis or inhibits tumor growth either invitro or in vivo in animals. For example, human test doses can beextrapolated from effective doses in cell studies, such as IC₅₀ values,or from effective doses in vivo by extrapolating on a body weight orsurface area basis. Such extrapolations are routine in the art. Further,one of skill would know how to formulate or prepare diets or dietarysupplements containing the analogs. In the case of animal diets, theanalogs could be added in concentrations up to 5% by weight and mixedaccording to methods routine in the art.

[0023] Dietary supplements for animals or humans could be prepared in avariety of forms that would include but not be limited to liquid,powder, or solid pill forms. Pill forms for the supplements would beprepared by methods routine in the art of dosage formulation and couldinclude but not be limited to production of gel capsules, time-releasecapsules, or solid pills formulated with excipients and binders. Again,one of skill in the art would know how to formulate the extracts orcompounds isolated therefrom based on its chemical nature and thedesired effect. The extract and/or the compounds isolated therefromcould also be administered topically in liquid or creme of lotion formsor by injection. Injectable forms would be prepared by solubilizing in apharmaceutically acceptable vehicle.

What is claimed is:
 1. A method for treating cancer by inducing Bcl-2phosphorylation in an animal, the method comprising administering to theanimal a composition comprising a sesquiterpene lactone in an amountsufficient to induce said phosphorylation to treat said cancer.
 2. Themethod of claim 1, wherein the composition consists essentially of10-acetylbritannilactone.
 3. The method of claim 1, wherein thecomposition consists essentially of 1,6-O,O-diacetylbritannilactone. 4.The method of claim 1, wherein said cancer is ovarian cancer.
 5. Themethod of claim 1, wherein said cancer is prostrate cancer.
 6. Themethod of claim 1, wherein said cancer is a breast cancer.
 7. The methodof claim 1, wherein the composition is administered to the animal aspart of a dietary supplement.
 8. The method of claim 1, wherein thecomposition is extracted from Inula britannica.
 9. The method of claim1, wherein the composition is extracted from Inula britannica floralparts.